1. Field of the Invention
The present invention relates to procedures for the determination and/or detection of immunologically reactive analytes such as ligands and ligand receptors. More particularly, the invention relates to methodology that is valuable for determining and/or detecting the presence of threshold levels of analytes such as therapeutic drugs, toxic materials, drugs of abuse and hormones and the like that are indicative of a physiological condition. In this latter regard, in one particularized aspect, the invention relates to procedures for detecting the presence of threshold levels of hormones such as progestin and estrogen derivatives and luteinizing hormone (LH) in human urine samples. The invention also relates to kits of materials for use in conducting such assay procedures.
2. Description of the Prior State of the Art
There has long been a need for measuring substances with a high degree of sensitivity and specificity. In particular, in fields such a clinical medicine, forensic science, environmental quality testing, food quality assurance, drug testing and other related areas, the presence and/or amount of trace substances in test samples is often of great significance. In such areas, the measurement of very low concentrations in the order of parts per million or less is often necessary. Moreover, such testing or measurement generally requires the identification of particular molecules while not sensing other molecules with similar yet different structures.
The need for sensitive and specific tests has been addressed in the past by the development of a number of immunoassay procedures based on the highly specific and sensitive interaction between an antigen and an antibody directed against such antigen. Antigens and antibodies are initially recognized as being the participants in the immune process of an animal, that is, when an animal is injected with a foreign substance that is an antigen, the animal in time responds by producing antibodies which are protein molecules that recognize and tightly bind the invading antigen thereby facilitating removal or destruction of the latter. The immune process is highly specific and the use of immunoassay procedures for identification of specific substances has been exploited with great success. Such procedures have been further facilitated by the important discovery of Milstein and Kohler reported in Nature 256: 495-497, 1975 which concerns the preparation of so-called monoclonal antibodies. The details of this work are well known and there is no need to repeat the same here; however, as a result of the Milstein and Kohler work, the development of highly sensitive and specific reagents has been particularly facilitated.
Known assay procedures include radioimmunoassay (RIA) procedures, enzyme immunoassays (EIA), enzyme linked immunosorbant assays (ELISA), fluorescent assays, chemiluminescence assays and assays wherein metal particles such as gold sol particles are used as tags or labels. These prior procedures are referred to and described in a commonly assigned, co-pending application Ser. No. 105,285, filed Oct. 7, 1987, the entirety of which is hereby specifically incorporated by reference.
Some of the known assays operate on the basis of a competitive immunoreaction. In performing competitive immunoassays, one generally mixes (1) a first immunoreactive substance (contained in an unknown sample), (2) a second immunoreactive substance that is specifically reactive with the first substance, and (3) a quantity of a third immunoreactive substance that has immunological reaction characteristics that are immunospecifically the same as the immunological reaction characteristics of the first immunoreactive substance. The third substance carries a detectable tag. During the course of the immunoreaction, the first and third substances compete with one another for binding sites on the second substance. After a predetermined time of immunoreaction, the second substance is separated and the amount of third substance bound thereto is determined. If the first substance is initially present at low levels, then the amount of third substance and therefore the amount of detectable tag bound to the second substance will be elevated. On the other hand, if the amount of first substance is elevated, then the amount of detectable tag bound to the second substance via the third substance will be low. Thus, at all levels, the amount of detectable tag which becomes bound to the second substance will be inversely proportional to the amount of first immunoreactive substance in the sample. At intermediate levels, the amount of detectable tag bound to the second substance is monotonically and inversely proportional to the level of the first immunoreactive substance in the sample.
Competitive immunoassays have found widespread use in clinical laboratories yielding accurate measurements of a great number of clinically relevant analytes. However, there are two features of the competitive assay format that are less than highly desirable outside of a sophisticated clinical laboratory setting. First, as discussed above, the resulting signal is inversely proportional to the amount of substance to be detected. In the ideal case, however, one would prefer that elevated levels of analyte should produce elevated levels of signal. Thus, in a non-instrumental, e.g. visual, examination, the amount of signal produced would be directly proportional to the amount of analyte detected. Secondly, in the competitive immunoassay format described above, the amount of detectable signal changes as a monotonically decreasing function of the amount of analyte in the sample. Thus, for analyte levels that are very close to one another, only modest shifts in signal intensity are produced. Such modest shift, although readily detectable with modern instrumentation, may often be too subtle for reliable detection by the human eye for direct visual examination purposes.
Where toxic and/or environmentally undesirable substances are concerned, once a non-hazardous threshold level has been selected as a criteria, it is advantageous to use tests which provide a completely negative result at levels even minutely below the threshold level and yet provide a clear positive indication when the level exceeds the selected threshold level.
In a more specific sense, there has long been a need for simple yet reliable methodology for determining the human fertile period during the menstrual cycle, that is, the period in which viable sperm and a viable ovum may both be present in the reproductive tract of the female. For a variety of reasons, contraceptive devices and materials may not be available for use, and accordingly, techniques for ascertaining the fertile period of the menstrual cycle have become desirable. Manifestly, techniques for ascertaining the fertile period of the menstrual cycle are valuable both for intentionally avoiding pregnancy and for facilitating conception when pregnancy is desired.
The menstrual cycle is governed by the release of hormones from the female glands and organs. Such release is predictable and specifically related to ovulation by which ova are released from the ovaries and the lining of the uterus is made ready for pregnancy and the hormones and/or metabolites thereof find their way into the urine. The specific biological phenomena are described in detail and with clarity in European Patent Publication No. 0086095, which was published on August 17, 1983 in European Patent Office Bulletin 83/33. And suffice it to say that during a normal menstrual cycle, the level of estrone-3-glucuronide (E.sub.1 3G) in female urine begins to rise about 6 days prior to ovulation and reaches its peak about 1 day before ovulation and falls rapidly during and after ovulation. The level of pregnanediol-3-glucuronide (P.sub.3 G) in female urine begins to rise on the day of ovulation, and reaches a peak 2 to 3 days after ovulation and remains elevated for the duration of the luteal phase. Likewise, the relationship between P3G and E.sub.1 3G levels is well known, and from the '095 European patent publication identified above, the ratio of estrogen to progestin metabolites in the urine has been found to be useful in monitoring the progress of the menstrual cycle.
Of particular importance in following the menstrual cycle by determining hormonal activity is the fact that approximately during ovulation, the level of P.sub.3 G in the urine surges to levels above 4 ug/ml. Thus, a simple and reliable assay capable of determining and/or detecting the presence of at least such threshold amount of P.sub.3 G in urine would be extremely valuable in determining whether ovulation has occurred.